Intrinsic and non-cell autonomous roles for a neurodevelopmental syndrome-linked transcription factor

Transcription factors (TFs) are essential for neuronal identity, yet their potential non-cell-autonomous functions remain largely unexplored. Here, we uncover both cell- and non-cell-autonomous roles for the conserved terminal selector UNC-3 in <i>C. elegans</i> motor neurons (MNs). UNC-3 is an ortholog of human EBF3, mutations in which cause a severe neurodevelopmental syndrome. Single-cell RNA sequencing of cholinergic MNs, which express <i>unc-3</i>, and downstream GABA MNs, which do not, revealed that <i>unc-3</i> loss disrupts neuronal identity in distinct ways across MN classes. Four cholinergic MN classes lose their molecular identity entirely, whereas the AS class retains it partially, illuminating terminal selector-driven neuronal diversification processes. Integrated transcriptomic and genomic analyses uncovered a dual cell-autonomous role for UNC-3 as both a direct activator and repressor of neuron-type-specific genes in cholinergic MNs, including repression of alternate neurotransmitter programs. Unexpectedly, <i>unc-3</i> loss also caused widespread transcriptional, morphological, and connectivity defects in downstream GABA MNs. Mechanistically, these non-cell-autonomous effects are mediated by cholinergic neurotransmission and include activation of the pro-regenerative bZIP TF CEBP-1 (C/EBP) and dysregulation of UNC-6/Netrin signaling. These findings redefine terminal selectors as both intrinsic and extrinsic regulators of neuronal identity and circuit assembly, offering a mechanistic framework for understanding EBF3 syndrome pathogenesis.